Synaptic vesicles are required for release of neurotransmitters and subsequent communication between neurons. The synaptic vesicle membrane proteins that facilitate fusion with the plasma membrane are transported from the cell body in synaptic vesicle precursors. These precursors must undergo a maturation process before they are functional synaptic vesicles and this process likely involves fusion with endosome-like compartments within the synaptic terminal. The Rab-5 GTPase is associated with canonical early endosomes, and known to be present in presynaptic terminals. Previous research has shown that biasing the cycling state of Rab-5 towards a primarily GTP-bound form likely leads to increased association of Rab-5 with endocytic membrane within the synaptic terminal, resulting in a concomitant decrease in the numbers of synaptic vesicles. To further understand the role of Rab-5 in neurons, I have conducted a forward genetic screen in C. elegans for mutations that disrupt YFP:: RAB-5 expression in the GABAergic motor neurons. Several classes of mutations have been isolated, and I have mapped and identified one mutation to the gene for Rabx-5, a predicted guanine exchange factor for Rab-5. The goals for this proposal are to test the hypothesis that Rabx-5 regulates endosomal and synaptic vesicle trafficking and transport in addition to functioning as the guanine exchange factor (GEF) for Rab-5 in C. elegans neurons and to continue to characterize other mutants revealed in this screen. Specific Aims: 1. Characterize the role of Rabx-5 in membrane and synaptic vesicle trafficking in developing neurons using mutant analysis of fluorescently marked synaptic vesicles and membrane compartments. 2. Map and characterize other molecules involved in regulation of Rab-5 compartments using single nucleotide polymorphism mapping and whole-genome sequencing. Because defects in regulation of the endocytic pathway and axonal transport contribute to neurodegenerative disease such as Alzheimer's disease, Huntington's disease, and fronto-temporal dementia, achieving a full understanding of the molecules and processes involved in the endocytic pathway and in axonal transport of endosomes is essential for for developing treatments for neurodegenerative diseases. RELEVANCE: This study will identify and characterize molecules involved in the "endocytic pathway" of neurons. Misregulation of the endocytic pathway contributes to neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and front-temporal dementia.